Gonadotropin-Independent Regulation of Steroidogenesis: MATERIALS AND METHODS(3)

4 Nov

Isolation, Purification, and Culture of Adult Leydig Cells

For isolation and purification of adult Leydig cells we used a previously described method . In brief, the testes were decapsulated carefully and incubated in 10 ml of culture medium for 10 min at 34°C in an atmosphere of 5% C02 in air in the presence of 0.3 |xg/ml collagenase type II (Sigma). After incubation, the tube (50 ml Falcon) was filled with medium and allowed to stand for 5 min at room temperature. The supernatant containing the dissociated interstitial cells was filtered through nylon gauze and centrifuged at 120 X g for 10 min. buy ampicillin

The resulting cell pellet was washed twice with the medium, and subjected to purification in a 50-ml continuous Percoll (Pharmacia, Uppsala, Sweden) gradient (density range 1.01—1.12 kg/L). After centrifugation (800 X g for 20 min at room temperature), the cell band containing the purest Leydig cells (density — 1.05 kg/L) was collected, washed with medium, and incubated in 24-well plates (105 cells in 1.0 ml medium/well). The purity of the Leydig cells was assessed by a cytochemical 3(B-HSD reaction after the Percoll fractionation, and 75-85% of 3(3-HSD-positive cells were found. The purified Leydig cells were allowed to attach for 24 h in an atmosphere of 5% C02 in air, after which the medium was changed and the cells were stimulated as described above.